Method of carrying out blood tests

ABSTRACT

The present invention relates to a method of carrying out blood tests. The invention is based on the finding that clinico-chemical blood parameters can be determined using not only blood serum but also blood plasma. Therefore, freshly taken blood samples, which are mixed with at least one thrombin inhibitor, can be used for determining both clinico-chemical parameters and hematological parameters. The clinico-chemical parameters can thus be determined using blood plasma, a separation of coagulable components being not required any more.

FIELD OF THE INVENTION

The present invention relates to a method of carrying out blood tests.

BACKGROUND OF THE INVENTION

Many diverse blood tests are required for diagnosing, in particular,internal diseases and for controlling the course thereof. These bloodtests can be divided into tests regarding the cellular blood components(blood count, differential blood count, blood group, immunophenotypingof blood cells) and into serum tests and also into antibody screeningtests, Coombs tests and cross-matching tests. Serum tests are concernedwith the determination of enzymes, metabolic products (e.g. creatinine,urea, blood sugar) and coagulation parameters. In addition, specialtests, such as hormone analyses or drug level analyses, are carried outon the basis of serum.

With seriously ill and hospitalized grown-up patients, enlarged bloodtests have to be carried out over a prolonged period of time every dayand sometimes even twice a day to detect critical changes in thephysical condition of the patient at an early stage. Blood is takenunder sterile conditions normally using a closed blood taking system bypuncturing a vein in the bend of the elbow (e.g. V. cubita mediana).Blood is optionally taken through already laid central indwellingcatheters. Depending on the clinical requirements and the desiredlaboratory tests, types and numbers of the tubes to be filled with bloodare defined. Specific laboratory tests can only be carried out withspecific substances specifically prepared for such tests (e.g. specificanticoagulants (heparin, EDTA, citrate, etc.)) or with glucosidaseinhibitor (“blood sugar tubes”) or blood withdrawing tubes containingcoagulation-promoting substances (“serum tubes”). Heparin and EDTA areunspecific, indirect inhibitors. They do not act directly orspecifically on a specific element of the blood coagulation cascade, buttheir effect is of an indirect nature in that they intercept, forexample, Ca²⁺ ions which, in turn, are essential for the activation ofdifferent proteins of the coagulation system. A uniform pretreatment ofthe blood to be tested (“standard tube”), on the basis of which all orat least the majority of important blood tests could then be carriedout, does not exist. Furthermore, it follows from the standard logisticsequence in clinico-chemical or hematological laboratories that aplurality of blood withdrawal tubes that have been pretreated in thesame way are often needed. After withdrawal from the patient, anddepending on the laboratory values to be determined, the blood tubes aretransferred into laboratories that are most of the time separatedspatially (most of the time a clinico-chemical laboratory and ahematological laboratory and optionally laboratories for special tests,such as immunophenotyping, drug level in the serum, etc. (materialdispatch by mail or courier might here be necessary). The blood tubesreceived in the laboratory must first be sorted, and the tubes to becentrifuged are then centrifuged at about 3,500 r.p.m. for five minutes.The blood taking tubes are then forwarded to the different work places(coagulation tubes to the coagulation place, serum tubes for electrolytedetermination on the flame photometer, etc). The respective automaticanalyzing devices are then loaded with the samples, with the samplevolume of about 10 μl to 100 μl, which is needed for one measurement,being very small in most measuring operations. The actual measuringoperation lasts from a few seconds to a few minutes (five minutes at themost, depending on the method and the device). The measured values arefinally printed out and, depending on the origin, are communicated inwriting to the dispatching stations.

In summary, it is necessary at the moment that several blood takingtubes (depending on the desired test) should be filled with 2-10 mlblood (depending on the tube size) in a blood taking process. Thefollowing tubes are needed for determining the routine laboratoryparameters:

blood count and differential blood count 1 × EDTA tube sodium, potassiumin the serum 1 × serum tube liver enzymes and/or 1 × serum tubecreatinine and/or urea and/or lipase and amylase and/or creatininekinase and/or cholesterol and triglycerides and/or lactate dehydrogenasetotal protein and protein electrophoresis 1 × serum tube glucose in theserum 1 × glucose tube Quick, PTT 1 × coagulation tube (citrate) bloodsedimentation rate 1 × BSR tube total 7 tubes

If, in addition, the blood group has to be determined and an antibodyscreening test has to be carried out and erythrocyte concentrates haveto be provided for, two further blood taking tubes (without additions)have to be taken. In the case of special tests that are required, e.g.hormone level analyses (T3, T4, TSH basal, etc.), drug level (digitoxinlevel, vancomycin level, theophylline level, etc.), special electrolyteconcentrations (magnesium, calcium, phosphate) and special coagulationvalues (deficiency in factors, fibrin degradation products), and manyothers, an additional withdrawal tube (most of the time serum tube) isneeded for each test as a rule.

Hence, independently of the blood taking system, the following seriousdrawbacks are found in these blood taking methods that have so far beenin general use:

1. The daily blood withdrawals which must be performed in the case ofseriously ill patients lead to a blood loss of about 250 ml per week.This is an amount approximately half the blood donation amount of ahealthy person at the German Red Cross. Another drawback is thatseriously ill persons often suffer from anemia caused by very differentfactors.

2. The many blood withdrawing operations that are required are aconsiderable cost factor in medical care; on the one hand, because ofthe purchasing costs and, on the other hand, because of the considerabledisposal costs for the tubes. The blood tubes used are classified asinfectious wet waste and must be burnt being packed in specialcontainers. In the case of seriously ill persons about 40 blood takingtubes are needed every week.

3. The workplace classification which is defined and segmented by thedifferently pretreated blood samples (anticoagulated whole blood forblood count determination, serum for enzyme tests and electrolytedeterminations, etc.) and by the automatic measuring devices adaptedthereto requires a multitude of work places entailing correspondinglyhigh costs with respect to personnel and financing.

SUMMARY OF THE INVENTION

It is the object of the present invention to provide a method forcarrying out blood tests, whereby the above-mentioned drawbacks can beovercome and many blood measuring parameters can be determined rapidlyand reliably within a short time interval almost at the same time.

This object is achieved by a method according to claim 1. Theachievement of such an object is, in particular, due to the finding thatalmost all of the clinico-chemical blood parameters can be determinedusing not only blood serum, but also blood plasma provided such bloodplasma—in contrast to standard practice—has been prepared by adding athrombin inhibitor, such as hirudin. The difference between serum andplasma is that the first one is free from fibrin whereas the latterstill contains fibrinogen. Surprisingly enough, it has been found thatone and the same blood sample can be used for determining bothclinico-chemical parameters and hematological parameters provided thesample is mixed with a thrombin inhibitor. Preferably, hirudin and/ordesulfatohirudin is/are used as thrombin inhibitor. This method can becarried out in an automated manner.

Hirudin is a highly specific thrombin inhibitor which is naturally foundin the salivary gland secretion of leeches, Hirudo medicinalis. Theanticoagulative activity in salivary gland secretions of Hirudomedicinalis was described by Haycraft for the first time about 100 yearsago (Haycraft, J. B. (1894), Naunyn-Schmiedebergs Arch. Exp. Pathol.Pharmakol. 18, 209). In the fifties Markwardt et al. succeeded inobtaining hirudin in pure form and in characterizing it biochemically(Markwardt, F. and Walsmann, P. (1958), Hoppe-Seyler's Z. physiol.Chemie 312, 85). Hirudin is a polypeptide of which various, naturallyoccurring variants have become known in the meantime and which has amolecular weight of about 7000 Dalton. Natural hirudin is used as athrombin inhibitor for biochemical studies (Walsmann et al., (1988)Pharmazie 43, 737). The use of hirudin has been limited to a very smallnumber of very special applications because of the small hirudin amountsfound in leeches and because of the troublesome extraction of hirudin.Among other things, hirudin has also been suggested for theanticoagulation of blood samples for determining the function and changein state of blood cells (e.g. blood sedimentation rate) (EP-A-442 843).

Since the end of the eighties it has been possible to preparedesulfatohirudin by genetic engineering techniques using yeasts orbacteria in great amounts. This recombinant desulfatohirudin isidentical with the natural hirudin of Hirudo medicinalis (except for amissing sulfate group on tyrosine 63) and has the same anticoagulantcharacteristics. On account of the production costs, which are stillhigh, recombinant hirudin is preferably developed for use in thetherapeutic field. Its yield, however, could be increased by the use ofnovel expression systems, such as the yeast Hansenula polymorpha, to anextent (Weydemann et al. (1995), Appl. Microbiol. Biotechnol 44, 377)that non-therapeutical applications present themselves for hirudin.Recombinant desulfatohirudin is easily soluble and can already inhibitblood coagulation in small concentrations. This makes it possible tosupply recombinant desulfatohirudin either in dissolved form or as a drysubstance and thereby to effectively inhibit blood coagulation. Theamount of the supplied desulfatohirudin can be dosed such that the bloodtaken remains incoagulable, depending on the time required bycorresponding tests. Furthermore, the amount of desulfatohirudin can beproportioned such that there will be no dilution effects on the bloodvolume to be taken and the measurement result will thus not beinfluenced. The observation of standard volumes is a critical factor inthe use of citrate solution as anticoagulant. In contrast to Na-EDTA orcitrate solution, the anticoagulating effect in desulfatohirudin is notdue to the withdrawal of Ca²⁺ ions, but to a specific steric thrombininhibition. As a result, no bivalent cations are removed from the blood,which permits an examination of cellular blood components underphysiological conditions. Moreover, desulfatohirudin does not requireany endogenous factors for its anticoagulative activity, as is e.g. thecase with heparins. For instance, heparin requires the factorsantithrombin III and heparin cofactor 2 for efficiently inhibitingcoagulation. With desulfatohirudin, blood samples that are derived frompatients suffering from a corresponding factor deficiency can beanalyzed without any additional measures and without any problems. Itcan be expected because of theoretical considerations that naturalhirudin variants and hirudin fragments, as far as they have athrombin-inhibiting effect, produce the same results as have beenobtained for recombinant desulfatohirudin. Furthermore, syntheticthrombin inhibitors can be used with the same result.

When hematological parameters are examined, the blood cells must bevital at the time of examination. The blood must be anticoagulated andmust not be changed in its volume parts by the course of theexamination; otherwise, the numerical values would be distorted.Hematological test parameters are, e.g., the number of erythrocytes,leukocytes and thrombocytes per volume unit of whole blood, theproportionate composition of the leukocytes from the various nucleatedblood cells which is determined under morphological criteria(differential blood), and the degree of loading of the individualerythrocytes with hemoglobin (quotient from the clinico-chemicalmeasurement value hemoglobin concentration and the number of individualerythrocytes per volume unit). Further hematological measurementparameters using immunological test reactants describe the surfaceproperty (antigenity) of intact erythrocytes (transfusion-serologicaltests, cross-matching of conserved blood) or mononuclear blood cells(immunopnehotyping for diagnosing e.g. malign blood diseases). All ofthese hematological tests have in common that they can only be carriedout on undestroyed, vital blood cells.

Hematological measurement parameters are determined either manually bymicroscope (e.g. by means of a counting chamber for determining thenumber of blood cells; blood smear preparation for assessing the cellmorphology (cell size, nucleus shape, cytoplasma characteristics,cytoplasmatic granulation, etc.)) or in automatic blood-cell countingdevices (e.g. Coulter counter). The determination of the hemoglobinvalue in blood is a clinico-chemical and not a hematological measurementparameter.

The present method is a method which can operate in an automated mannerwith whole human blood in a blood taking tube in a novel manner and insuch an anticoagulated fashion that the blood sample can simultaneouslybe used for the automated determination of hematological,clinico-chemical and immunological measurement parameters, i.e. inshort, for the determination of almost all measurement parameters. Theroutine measurement methods which have so far been in use can beemployed entirely or after having been slightly adapted to the novelanticoagulant.

Furthermore, it should be noted that a quantitative determination bymeans of automatic measuring devices is possible according to thepresent method. Fresh capillary blood is used for a manualdetermination, e.g. in the counting of blood corpuscles. In largelaboratories hematological tests, however, can no longer be carried outmanually, but only by automated measuring devices. For this purposevenous K2-EDTA-anticoagulated whole blood is resorted to. Thanks to theaddition of the anticoagulant dipotassium-ethylenediaminetetraaceticacid (K2-EDTA) in the worldwide standard concentration of 1 mg per 1 mlof blood, a high concentration of potassium ions is introduced into theblood sample, apart from the desired complexation of the calcium ions bythe EDTA. Such an artificially high potassium concentration effects achange in the osmotic gradient for potassium between the leukocytes(granulocytes and lymphocytes) and the ambient aqueous environment, withthe effect that the leukocytes lose cell water and shrink. After sometime (30 minutes) a new osmotic gradient for potassium ions is obtainedthrough compensation mechanisms of the leukocytes, and the cells recoverpart of the lost cell water. This new equilibrium remains stable forseveral hours. Of course, it is different in comparison with the nativeblood which has no K2-EDTA added thereto. It is only when the nucleatedblood cells in K2-EDTA-anticoagulated blood have been adapted to thisnew equilibrium and have reached a shrunken, but stabilized form, thatthey can be counted in repeatable form in automated measuring devicesand can reliably be distinguished from one another in volume,conductivity and stray-light characteristics (automated differentialblood counting). An effect similar to the K2-EDTA sample pretreatment isnot observed with hirudin, which seems to rule out the use of hirudinblood for automated measurements. Surprisingly enough, however, thepresent results show that quantitative hematological measurementparameters can be determined in automated measuring devices withhirudin-anticoagulated blood.

When hematological routine parameters, such as the number oferythrocytes, leukocytes and thrombocytes, are determined in anautomated manner, the anticoagulants sodium citrate, sodium oxalate andheparin lead to wrong measurement results, which is generally known. Theaddition of sodium citrate or sodium oxalate to whole human blood willshrink the erythrocytes to such a considerable extent that the situationprevailing in the non-anticoagulated native blood cannot be inferredfrom the automated size determination of the erythrocytes and thecalculated results, e.g. hematocrit. Moreover, when sodiumcitrate-anticoagulated blood is used, the numerical value of all of theblood cells measured and of the hemoglobin value must be corrected bythe factor 1.1. What is even more disadvantageous is the fact that thenumerical values of the blood cells vary at random, which could so farnot be explained in a satisfactory manner, with the variations having arange of 10% in the case of thrombocytes and, in pathologically reducedthrombocyte values, even a greater range. When heparin-anticoagulatedblood is used, unforeseeable, random spontaneous aggregations ofthrombocytes and leukocytes may occur, so that during counting inautomatic measuring devices partly falsely low measured values aredetermined for these cells. When anticoagulants, such as heparin,calcium citrate, but also K2-EDTA, are used, such measurement errors areabove all observed in the determination of hematological parameters whenduring the automatic differentiation in the hydrodynamically focusedsample flow, the leukocytes are differentiated according to volume(resistance measurement in direct current), conductivity (measurement ofthe internal conductivity of the cells with high-frequency alternatingcurrent) and stray-light characteristics (measurement of the typicalsurface structures of the cells and their peripheral granulation with ahelium-neon laser) (automated differential blood counting). Forinstance, a heparin addition during automatic differential bloodcounting is, in particular, detrimental to an exact recognition of thebasophilic granulocytes which are rather rare, but particularlyimportant from a diagnostic point of view. Typically, an excessivelyhigh value of basophilic granulocytes is indicated. Moreover, such awrongly determined blood count based on the heparin-anticoagulated wholeblood is difficult to be checked manually, such a check, however, beingimperative. On account of its great molecular electrostatic charge, theheparin addition interferes with the necessary stains (May-Grünwaldstain and Giemsa stain) with which the blood cells which have beensmeared on an object carrier of glass are fixed and stained (panopticstaining according to Pappenheim). Thus, the addition of heparin effectsa blue tinge of the nucleated blood cells, which makes it difficult oreven impossible to distinguish the blood cells by microscope. Finally,serious measurement errors may even be caused by the K2-EDTA which isrecommended and used worldwide for determining hematological routineparameters in automated measuring devices, but also in the case ofmanual determinations. It often happens that small blood clots areformed for the reason that the K2 EDTA-coated blood tubes are notimmediately tilted and moved after the withdrawal of blood. Such partlyclotted blood samples must not be processed, for they lead to wrongnumerical values of the blood cells in the case of determiningoperations carried out by machine or manually. Moreover, they might clogthe microcapillaries of the automatic measuring devices.

Since these many undesired interferences of the different substances forthe anticoagulation of whole human blood have been known and could notbe foreseen in detail upon the introduction of such substances, it couldbe assumed that hirudin-anticoagulated whole blood would also interferein a disadvantageous manner with some of the important routinemeasurement methods. Therefore, there was some general prejudice amongthe experts that the determination of a multitude of clinico-chemicalparameters and hematological parameters on the basis of a single bloodwithdrawal vessel is not possible.

With the method according to the invention, however, all of the relevantclinico-chemical and hematological values can be determined from asingle blood withdrawal container at the same time. Possible blood valuedeterminations comprise:

1. Determination of the serum parameters (e.g. alkaline phosphatase,amylase, cholinesterase, creatine kinase, GOT, GPT, γ-GT, HBDH, lactate,LDH, lipase, albumin, bilirubin, calcium, chloride, cholesterol,creatinine, iron, total protein, glucose, uric acid, urea, potassium,magnesium, sodium, triglycerides, C-reactive protein, immunoglobulins,transferrin, anti-streptolysin-O, rheumatoid factors, C3, C4,apolipoprotein, drug level). A determination is preferably carried outin automated measuring devices after separation of the corpuscularcomponents from the whole blood. For a further simplification of themethod the serum parameters can also be determined in automatedmeasuring devices in which a separation of the corpuscular components isno longer required.

2. Examination of the blood count: partial blood count (automated) anddifferential blood count (manual and automated).

3. Determination of the blood group, carrying out antibody screeningtests, carrying out Coombs tests and cross-matching erythrocytesconcentrates.

4. Immunophenotyping of normal and malign mononuclear cells in blood andbone marrow.

A blood withdrawal tube (universal standard tube) which can be used forthe present invention may be a blood withdrawal vessel that contains thethrombin inhibitor as a solution or as a dry substance or as a surfacecoating. The amount of the thrombin inhibitor should be proportionedsuch that for a period of time in which all of the above-describedanalyses can be carried out, the coagulation of the withdrawn bloodvolume is fully inhibited, thereby permitting a conduction of themeasurements in the illustrated method without any problems. Forinstance, tubes, syringes, pipettes and capillaries of plastic material,glass or metal are suited as blood withdrawal vessels.

A further embodiment of the present invention is concerned with theperformance of the blood test with automated measuring devices thatcombine the following functions in one unit:

determining all clinico-chemical parameters,

determining all hematological parameters,

wherein in the course of the measuring operation first the hematologicalvalues (blood count/differential blood count) should be recorded andthen, following a selective removal of the cellular blood components(=hirudin plasma), the clinico-chemcial parameters (including specialdeterminations) should be determined. The cellular components can e.g.be removed via a microfilter or by centrifugation. Furthermore, theabove-described blood withdrawal vessels can also be used with measuringdevices which carry out determining operations by means of test strips.

It is a further object of the present invention to provide a method ofdetermining clinico-chemical blood parameters which can be carried outin a more simple and faster manner than known methods.

This object is achieved by a method set forth in claim 10. Surprisinglyenough, it has been found that a freshly taken blood or bone marrowsample which is mixed with a thrombin inhibitor can be used fordetermining the above-mentioned clinico-chemical parameters. Aseparation of fibrinogen is not necessary. Preferably, however, thecellular and corpuscular components, as described above; can beseparated from the blood plasma prepared in this manner.

It is advantageous to use hirudin and/or desulfatohirudin, preferablyrecombinant hirudin and/or desulfatohirudin, as the thrombin inhibitor.

In this method, too, the freshly taken blood sample should be put into awithdrawal vessel in which the thrombin inhibitor is provided.

The clinico-chemical parameters can be determined by means of anautomated measuring device.

DETAILED DESCRIPTION OF THE INVENTION

The present invention shall be explained in more detail with referenceto the following examples:

EXAMPLE

1. It could be shown that the use of recombinant hirudin of the companyRhein Biotech, Düsseldorf, made whole human blood incoagulable in bloodtaking tubes for differently long periods of time in proportion to thehirudin amount used. At a concentration of 200 ATU rhirudin (=20 ngrhirudin) per ml whole blood the blood was incoagulable for 24 hours; ata concentration of 100 ATU rhirudin (=10 ng rhirudin) the whole bloodclotted entirely in glass or plastic withdrawal tubes already after 12hours (see Table 1).

TABLE 1 Recombinant hirudin as an anticoagulant of whole human bloodCoagulation in hours after addition of the anticoagulant to the freshwhole blood of healthy test persons 0.5 test persons 2 4 8 12 16 20 2448 Hirudin in S J H S J H S J H S J H S J H S J H S J H S J H S J HATU/ml P R M P R M P R M P R M P R M P R M P R M P R M P R M 0 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 10 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 50 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 75 − −− + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +100 − − − − −− + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +150 − − − − − − − − − − + − − + − + + + + + + + + + + + + + + + + 200 −− − − − − − − − − − − − − − − − − − − − − − − + + − 250 − − − − − − − −− − − − − − − − − − − − − − − − − + − 500 − − − − − − − − − − − − − − −− − − − − − − − − − − − 750 − − − − − − − − − − − − − − − − − − − − − −− − − − − Heparin − − − − − − − − − − − − − − − − − − − − − − − − − − −5 IE/ml

Directly after withdrawal, 2 ml whole blood from three healthy testpersons (SP, JR, HM) was introduced into non-prepared glass tubes of theVacutainer blood withdrawal system (company Becton and Dickinson) whichhad previously been loaded with different amounts of recombinanthirudin. The stock solution of the recombinant hirudin was 10⁴ ATU perml, so that between 20 μl and 150 μl of the stock solution were used pertube. 5 IE/ml heparin was used as a positive control. The tubes werepermanently moved on a roller. The time up to the occurrence of smallclots having a diameter of about 1-2 mm (beginning coagulation (+)) andthe time up to the occurrence of a complete coagulation (++) wasmeasured. The presence of microclots inside the blood tubes, with themicrodots being unfavorable for the cell-number measuring operation anddistorting the result, was checked by repeatedly counting the cells bymeans of the STKS device of the company Coulter. The detection ofmicroclots was evaluated as a beginning coagulation (+). In cases wereno macro- or microclots were detectable, the anticoagulated blood couldbe analyzed in the STKS of Coulter without any problems (−).

2. It was shown that after centrifugation of the corpuscular elements amultitude of clinico-chemical laboratory assays could be carried out onautomated measuring devices without any problems and in a repeatablemanner on the basis of blood withdrawal tubes of glass that had beenloaded with 200 ATU rhirudin per ml blood. The measured values whichwere determined and existed in the pathological as well as in thestandard range were statistically not significantly different from thevalues obtained with the conventional method (serum method). Technicalproblems on the measuring devices, such as clogging of the suctioncapillaries, etc., occurred seldom and not more frequently than in theserum method. The following measurement parameters were determined in anautomated manner: GOT, GPT, alkaline phosphatase, gamma-GT, sodium,potassium, creatinine, urea, creatine kinase, bilirubin, lactatedehydrogenase, alpha-HBDH, amylase, lipase, glucose, total cholesterol,triglycerides, chloride, magnesium, phosphate, calcium, iron, totalprotein, protein electrophoresis, antistreptolysin titer, C-reactiveprotein, beta-HCG (see Table 2).

TABLE 2 test person M parameter serum hirudin plasma unit GOT/ASAT 9 9U/l GPT/ALAT 10 10 U/l AP 103 100 U/l GGT 10 10 U/l HBDH 96 98 U/l totalbilirubin 12.7 13 μmol/l CK 60 59 U/l lipase 79 82 U/l LDH 166 167 U/lamylase 75 73 U/l creatinine 94 88 μmol/l urea 6.6 6.4 mmol/l totalcholesterol 4.7 4.7 mmol/l triglycerides 1.6 1.6 mmol/l glucose 5.3 5.5mmol/l calcium 2.22 2.19 mmol/l magnesium 0.7 0.73 mmol/l phosphate 1.21.1 mmol/l iron 20 20.1 μmol/l potassium 4.2 3.8 mmol/l sodium 142 141mmol/l chloride 106 108 mmol/l osmolality 276 278 mosmol/kg totalprotein 76 77 g/l electrophoresis albumin 70.6 67.3 % alpha 1 2.2 2.1 %alpha 2 5.3 5.8 % beta 9.5 11.2 % gamma 12.4 13.6 % with gradients! AST<80 120 kU/l CRP <6 <6 mg/l HCG <2 <2 U/l

3. It was shown that, on the basis of the same hirudin-treated bloodtaking tubes of glass, a partial blood count (measurement andcalculation of the cellular amounts in the whole blood) couldadditionally be performed on automatic measuring devices and adifferential blood count could be made on automatic devices and by hand.The measured values which were found and which were within thepathological and the normal range were statistically not significantlydifferent from the values obtained in the routine method (EDTA blood).Technical problems on the automated cell counting devices (Coulter,USA), such as clogging of the suction cannula or R-alarm (registrationerror), did not occur often and not more frequently than in the standardmethod (EDTA blood). For a correct automatic determination of thedifferential blood count of rhirudin-anticoagulated whole blood,however, the automatic measuring devices which are set toEDTA-anticoagulated blood must be adjusted accordingly to the use ofrhirudin-anticoagulated blood. Granulocytic cells, in particular, shrinkand thus change due to the EDTA addition within the first 60 minutes.This has the effect that the determination of the automated differentialblood count of EDTA blood, e.g. on the STKS device of Coulter, will onlybe reliable if prior to testing the blood is EDTA-anticoagulated for aperiod of at least about 30 to 60 minutes. This measure which isnecessary because of the EDTA-induced unspecific interception of thefree calcium ions can be dispensed with in the highly specificanticoagulation by means of rhirudin, which anticoagulation is lessdetrimental to the cells. The morphology of the nucleated cells in thehand-counted differential blood count was better preserved inrhirudin-anticoagulated blood than in the EDTA blood, in particularafter a period of several hours.

The following test was performed:

Venous blood was taken from the cubital vein of a healthy test person(XM) and put into glass-made blood withdrawal tubes of the Vacutainerblood taking system (Becton and Dickinson). These tubes, which were notprepared by the manufacturer and were intended for the recovery ofserum, had previously been loaded with heparin (5 IE/ml) or recombinanthirudin (200 ATU/ml). An EDTA blood count tube (Becton and Dickinson)which was also filled with venous native blood of the test person servedas a control.

Four hours after the blood withdrawing operation (average transportationand dispatch time), the blood samples were evaluated by machine on theSTKS of Coulter. The EDTA blood was additionally evaluated immediatelyafter withdrawal. A partial blood count (numerical values) and adifferential blood count were carried out by machine. Furthermore, smearpreparations were made and the differential count was carried outmanually under a microscope on the following day.

In summary, the blood samples could be evaluated on an equally goodbasis independently of the anticoagulant used. There were no essentialdifferences in the numercial values in the partial blood count and inthe manually performed differential blood counts. However, as regardsdifferential blood counting by machine, a wrong measurement of part ofthe neutrophilic granulocytes, which were either recognized asbasophilic granulocytes or as monocytes, could be observed, as expected,on the STKS device of Coulter which had been calibrated to EDTA blood,namely in the case of both heparin blood and hirudin blood. This wrongmeasurement is equally found in the known manner when EDTA blood isevaluated by machine within the first hour after blood drawing. Thismeasurement error could be corrected by a corresponding operation.

4. Moreover, it could be shown that on the basis of the samerhirudin-treated blood withdrawal tubes of glass, and in addition to thealready described tests, the blood group could be determinedserologically, an antibody screening test could be performed, the Coombstests could be carried out and erythrocyte concentrations could betested. These tests could be carried out reliably and reproducibly.Technical problems did not arise. It should be emphasized that antibodyscreening tests, in particular, should be carried out with EDTA-freeblood, if possible, to avoid interferences with complement-dependentantibodies, such as Kit a, Kit b, Lewis a and Lewis b.

The future in carrying out antibody screening tests and cross-matches ofconserved blood, which are at the moment predominantly made manually onthe basis of clotted blood by means of dropping techniques and byvisually reading the agglutination, lies above all with large bloodbanks and hospitals in the automation of the pipetting and readingoperations. Since in the case where conserved blood is cross-matched,both serum or plasma and erythrocytes are needed from the patient, thepatient's blood must be anticoagulated and centrifuged when thecross-matching is to be carried out in an automated manner by means ofan automatic pipetting device. Otherwise, blood clots would be aspiratedupon removal of the centrifuged erythrocytes and the test would have tobe interrupted. It is true that anticoagulation can be carried out withEDTA. EDTA-anticoagulated blood, however, is only suited to a certaindegree for the performance of antibody screening tests, because EDTAinteracts with complement, and complement-dependent antibodies such asKit a and b, Lewis a and b might then no longer be detectable. Bycontrast, it could be shown for the first time that withrhirudin-anticoagulated blood, it was possible to carry out antibodyscreening tests and the cross-matching of blood in an automated mannerand without any problems with the help of the automatic pipetting deviceID-Sampler II using the Micro Typing Systems (both DiaMed AG,Cressier-sur-Morat, Switzerland).

The following tests could be carried out:

A: Manual Blood Group Typing and Manual Antibody Screening Test

5 ml venous whole blood from a healthy test person was introduced into ablood withdrawal tube of glass (Becton and Dickinson) which wasuntreated and had previously been loaded with recombinant hirudin (200ATU/ml). After 30 minutes the blood in the untreated glass tube wascompletely clotted, whereas the hirudin-treated blood wasanticoagulated. Both glass tubes were then centrifuged. Subsequently,the blood group was determined in both glass tubes, each timeserologially by means of a manual dropping technique and visuallyreading the agglutination. Moreover, antibody screening tests wereequally carried out with an antibody panel (screening) in threedifferent media (0.9% NaCl, bromelin and Coombs).

In summary, rhirudin-anticoagulated blood could be used for determiningthe blood group and for antibody screening tests in the same manner asclotted blood (standard method).

B: Automatic Performance of Antibody Screening Tests by Means of theAutomatic Pipetting Device ID-Sampler II Micro-Typing Systems, CompanyDiaMed AG, Cressier-sur-Morat, Switzerland

5 ml venous whole blood from a healthy test person was introduced into ablood withdrawal tube of glass (Becton and Dickinson) which hadpreviously been loaded with recombinant hirudin (200 ATU/ml). Theanticoagulated blood was centrifuged in the glass tube. The centrifugedblood withdrawal tubes was then put into the automatic pipetting deviceID-Sampler II of DiaMed. In this automatic pipetting device plasma wasthen taken from the blood tube and pipetted into the corresponding MicroTyping Cards of DiaMed-ID, which were prepared for carrying out anantibody screening test in NaCL-, Coombs and cold media. These MicroTyping Cards were then incubated at 37° C. and subsequently centrifuged.For an exact identification of the samples the bar codes on the MicroTyping Cards were then entered manually by means of a bar code readerinto a Compacq 4/50 PC. Finally, the Micro Typing Cards were put intothe ID Reader M of DiaMed, agglutination was evaluated photoopticallyand printed out via the computer.

In summary, it could be shown that rhirudin-anticoagulated whole bloodin the automatic pipetting device ID-Sampler II Micro Typing Systems ofDiaMed could be used without any problems for automatically carrying outantibody screening tests.

Finally, it could be shown that the immunophenotype of normal and malign(leukemia, lymphoma) mononuclear cells will not change in human blood orbone marrow if the testing material is, in contrast to standardprocedure, not anticoagulated by the addition of heparin, but isanticoagulated by the addition of rhirudin. The cell morphology in therhirudin-anticoagulated blood or bone marrow was considerably betterthan in heparin blood (standard method) even after a storage time of 96hours at room temperature (simulation of the transportation route incase of dispatch).

The following test was performed:

For immunophenotyping bone marrow blood (A) or peripheral blood (B), 5ml material was respectively aspirated into a plastic syringe preparedwith heparin (5 IE/ml) or recombinant hirudin (200 ATU/ml). Patient A issuffering from a carcinoma of the rectum. The test with his bone marrowhad been carried out because of the suspected additional presence ofacute myeloid leukemia. Patient B is suffering from chronic myeloidleukemia in a chronic phase. Testing on the basis of the peripheralblood was carried out for confirming the diagnosis. One hour (A) orthree days (B) (simulation of the transportation route in the case ofdispatch material, a frequent situation) after anticoagulabon at roomtemperature the material was centrifuged by a Ficoll gradient (companyPharmacia) and the mononuclear cells were thus separated. Themononuclear cells were then transferred by means of a pipette into aglass tube, the number of cells was determined on the Coulter counterand adjusted with 0.9% NaCl to a cell number of 5×10⁵. Various primaryantibodies were then pipetted to obtain fractions of this cellsuspension. Following incubation and washing of the cell suspension asecondary fluorescence-labeled antibody (FITC-conjugated F (ab′)₂ goatanti-mouse) was used for detecting the cell membrane-bound primaryantibody (indirect immunofluorescence). The cell suspensions which hadbeen treated with antibodies were then analyzed by flow cytometry(FACScan®, Becton and Dickinson). The numerical values correspond to thepercentage of the fluorescent cells which were evaluated.

In summary, it becomes evident that both heparin-anticoagulated blood(standard method) and hirudin-anticoagulated blood can be analyzedimmediately and also after three days in an equally good manner byimmunophenotyping by means of FACScan®, and that such an analysis leadsto comparable results, independently of the anticoagulation.

In summary, it could be shown for the first time that with the help ofrhirudin-anticoagulated blood from a blood withdrawal tube a multitudeof clinico-chemical routine and special tests (using the plasma) andalso blood group-serological, cytomorphological and quantitative bloodcell determinations (using the whole blood) are possible on an equalbasis with the standard routine methods. In practice, the followingpossible improvements can be inferred therefrom:

1. Blood loss, in particular in seriously ill patients, which is causedby frequent diagnostic blood withdrawals, sometimes several times a day,can be considerably reduced.

2. Costs can considerably be reduced, due to the low purchasing anddisposal costs of the blood withdrawal tubes and above all because ofthe possible integration of different diagnostic measurement and workplaces in laboratories with uniformly anticoagulated blood.

What is claimed is:
 1. A method of determining clinico-chemical andhematological parameters on blood, comprising the steps of mixing afreshly taken blood sample with at least one direct, specific thrombininhibitor and using said blood sample for determining bothclinico-chemical parameters and hematological parameters, wherein theclinico-chemical parameters are selected from the group consisting ofglutamine-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase(GPT), alkaline phosphatase, amylase, lipase, γ-glutamyl transferase(GGT), lactate dehydrogenase (LDH), creatine kinase, liver enzymes,cholinesterase, α-hydroxybutyrate dehydrogenase (α-HBDH); creatinine,urea, uric acid, bilirubin, glucose, total cholesterol, triglycerides;sodium, potassium, chloride, magnesium, phosphate, calcium; coagulationparameters C3, C4 and quick prothrombin time; β-human chorionicgonadotropin (β-HCG), T3, T4, and TSH basal hormones; iron, transferrin,hemoglobin, total protein, protein electrophoresis, albumin, C-reactiveprotein, immunoglobulins, antistreptolysin titer, rheumatoid factors,apolipoprotein, digitoxin, vancomycin, theophylline, and osmolality, andthe hematological parameters are selected from the group consisting oferythrocyte, leukocyte and thrombocyte counts, leukocyte fraction ofnucleated blood cells (differential count), antigenicity of blood cells,immunophenotyping of mononuclear cells, antibody screening tests, Coombstest, and blood sedimentation rate.
 2. A method according to claim 1,wherein hirudin, desulfatohirudin or both are used as the at least onethrombin inhibitor.
 3. A method according to claim 2, whereinrecombinant hirudin, desulfatohirudin or both are used.
 4. A methodaccording to claim 1 wherein cellular and corpuscular components areremoved from the blood sample prior to the determination of theclinico-chemical parameters.
 5. A method according to claim 1, whereinthe hematological parameters comprise erythrocyte, leukocyte andthrombocyte counts, or differential blood count or both.
 6. A methodaccording to claim 1, wherein the hematological parameters compriseantigenicity of blood cells, antibody screening tests, Coombs tests orany combination thereof.
 7. A method according to claim 1, wherein oneof the hematological parameters comprises an immunophenotyping operationwhich is carried out with the freshly taken blood sample obtained fromperipheral blood or bone marrow blood.
 8. A method according to claim 1,wherein the freshly taken blood sample is put into a containercontaining the at least one thrombin inhibitor.
 9. A method according toclaim 1, wherein the parameters are determined in automated measuringdevices.
 10. A method for determining clinico-chemical blood parameters,comprising the steps of mixing a freshly taken blood sample with ananticoagulant consisting essentially of at least one specific, directthrombin inhibitor and determining the clinico-chemical parameters usingthe resulting blood sample, wherein said clinico-chemical bloodparameters are selected from the group consisting ofglutamine-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase(GPT), alkaline phosphatase, amylase, lipase, γ-glutamyl transferase(GGT), lactate dehydrogenase (LDH), creatine kinase, liver enzymes,cholinesterase, α-hydroxybutyrate delydrogenase (α-HBDH); creatinine,urea, uric acid, bilirubin, glucose, total cholesterol, triglycerides;sodium, potassium, chloride, magnesium, phosphate, calcium; coagulationparameters C3, C4 and quick prothrombin time; β-human chorionicgonadotropin (β-HCG), T3, T4, and TSH basal hormones; iron, transferrin,hemoglobin, total protein, protein electrophoresis, albumin, C-reactiveprotein, immunoglobulins, antistreptolysin titer, rheumatoid factors,apolipoprotein, digitoxin, vancomycin, theophylline, and osmolality. 11.A method according to claim 10, wherein cellular and corpuscularcomponents are removed from the blood sample prior to the determinationof the clinico-chemical parameters.
 12. A method according to claim 10,wherein hirudin, desulfatohirudin or both are used as the at least onethrombin inhibitor.
 13. A method according to claim 12, whereinrecombinant hirudin, recombinant desulfatohirudin or both are used. 14.A method according to claim 10, wherein the freshly taken blood sampleis put into a container containing the at least one thrombin inhibitor.15. A method according to claim 10, wherein the clinico-chemicalparameters are determined in automated measuring devices.
 16. A methodaccording to claim 10 wherein the clinico-chemical blood parameters aredetermined using blood plasma.
 17. A method of using blood withdrawalcontainers containing at least one direct, specific thrombin inhibitor,comprising collecting blood in said containers and determining bothclinico-chemical and hematological parameters, wherein theclinico-chemical parameters are selected from the group consisting ofglutamine-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase(GPT), alkaline phosphatase, amylase, lipase, γ-glutamyl transferase(GGT), lactate dehydrogenase (LDH), creatine kinase, liver enzymes,cholinesterase, α-hydroxybutyrate dehydrogenase (α-HBDH); creatinine,urea, uric acid, bilirubin, glucose, total cholesterol, triglycerides;sodium, potassium, chloride, magnesium, phosphate, calcium; coagulationparameters C3, C4 and quick prothrombin time; β-human chorionicgonadotropin (β-HCG), T3, T4, and TSH basal hormones; iron, transferrin,hemoglobin, total protein, protein electrophoresis, albumin, C-reactiveprotein, immunoglobulins, antistreptolysin titer, rheumatoid factors,apolipoprotein, digitoxin, vancomycin, theophylline, and osmolality, andthe hematological parameters are selected from the group consisting oferythrocyte, leukocyte and thrombocyte counts, leukocyte fraction ofnucleated blood cells (differential count), antigenicity of blood cells,immunophenotyping of mononuclear cells, antibody screening tests, Coombstest, and blood sedimentation rate.
 18. The method of claim 17, whereinthe blood withdrawal containers are coated with said at least onethrombin inhibitor.
 19. A method of using blood withdrawal containerscontaining an anticoagulant consisting essentially of at least onedirect, specific thrombin inhibitor, comprising collecting blood in saidcontainers and determining clinico-chemical parameters, wherein theclinico-chemical parameters are selected from the group consisting ofglutamine-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase(GPT), alkaline phosphatase, amylase, lipase, γ-glutamyl transferase(GGT), lactate dehydrogenase (LDH), creatine kinase, liver enzymes,cholinesterase, α-hydroxybutyrate dehydrogenase (α-HBDH); creatinine,urea, uric acid, bilirubin, glucose, total cholesterol, triglycerides;sodium, potassium, chloride, magnesium phosphate, calcium; coagulationparameters C3, C4 and quick prothrombin time; β-human chorionicgonadotropin (β-HCG), T3, T4, and TSH basal hormones; iron, transferrin,hemoglobin, total protein, protein electrophoresis, albumin, C-reactiveprotein, immunoglobulins, antistreptolysin titer, rheumatoid factors,apolipoprotein, digitoxin, vancomycin, theophyllin, and osmolality. 20.The method of claim 17 or 19, wherein the at least one thrombininhibitor is selected from the group consisting of hirudin,desulfatohirudin and both hirudin and desulfatohirudin.
 21. The methodof claim 19, wherein the at least one thrombin inhibitor is selectedfrom the group consisting of recombinant hirudin, recombinantdesulfatohirudin and both recombinant hirudin and recombinantdesulfatohirudin.